One of the major causes for deafness is the loss of inner ear hair cells, the sensory cells that detect sounds. In mammals, hair cells are born during embryonic development and are maintained in quiescence throughout life. Mammalian inner ear, unlike the counterpart in lower vertebrates such as chick or fish, does not regenerate hair cells after damage. Deletion of negative growth genes (Rb1 and p27kip1) led to cell cycle re-entry in embryonic and neonatal inner ear. However, we have shown in adult inner ear, Rb1 deletion is not sufficient to induce proliferation. Further, the proliferating hair cells and supporting cells will ultimately die. Thus the inability to re-enter cell cycle by mature inner ear and apoptosis of proliferating cells present two main challenges to hair cell regeneration. This proposal is designed to specifically address the two issues. We showed that FGF signaling is necessary for hair cell regeneration in zebrafish neuromasts. For the specific aim 1, we will test the hypothesis that, with an inducible mouse model, FGF activation with Rb1 deletion could lead to cell cycle re-entry in adult inner ear in vivo. We have observed such events in vitro. Correlation of FGF activity in the proliferating cells by immunostaining will support the crucial role of FGF in cell cycle re-entry. Using an inducible mouse model to mark supporting cells genetically, we will identify hair cells derived from supporting cell transdifferentiation. In the aim 1b, we will characterize regenerated hair cells in differentiation, synapse formation and function by immunostaining, FM1-43 uptake and transduction current recording. In the second aim, we will evaluate two pathways, IGF1 and p53, for their roles in survival and apoptosis of proliferating hair cells. We have the evidence that IGF1 activation or p53 blockade protect Rb1-/- cochlear hair cells from apoptosis. In the aim 2a, we will determine the necessity of IGF1 in Rb1-/- cochlear hair cell survival by blocking IGF1 function to induce apoptosis. Further, we will specifically block two IGF1 signaling pathways: PI3K/Pdk1/Akt and Raf/Mek/Erk, to assess their respective role in the survival of Rb1-/- cochlear hair cells. In the aim 2b, we will block p53 function by a specific inhibitor and correlate p53 inactivation with Rb1-/- cochlear hair cell survival. We will further study the effects on the p53 pathway after IGF1 activation or inhibition. Inactivation of the p53 pathway after IGF1 activation, or vice versa, is an indication that IGF1 antagonizes p53 function to promote survival. Finally we will induce cell cycle re-entry in the adult inner ear on the p53-null background, and study long-term survival of proliferating hair cells. Cell cycle re-entry in mature inner ear and the survival of proliferating hair cells will make it possible to regenerate functional hair cells. PUBLIC HEALTH RELEVANCE: The proposal is designed to demonstrate that FGF activation and deletion of retinoblastoma gene can lead to cell cycle re-entry and regeneration of hair cells, the sensory cells that detect sounds, in adult mouse inner ear in vivo. It will demonstrate that manipulation of IGF1 (insulin-like growth factor) and p53 could promote survival of regenerated hair cells. Success of the proposal will lay the foundation for the study of functional recovery of hearing by regenerated hair cells.